Nonisotopic detection systems have become a preferred mode in scientific research and clinical diagnostics for the detection of biomolecules using various assays including, but not limited to, flow cytometry, nucleic acid hybridization, DNA sequencing, nucleic acid amplification, immunoassays, histochemistry, and functional assays involving living cells. In particular, while fluorescent organic molecules such as fluoroscein and phycoerythrin are used frequently in detection systems, there are disadvantages in using these molecules in combination. For example, each type of fluorescent molecule typically requires excitation with photons of a different wavelength as compared to that required for another type of fluorescent molecule. However, even when a single light source is used to provide a single excitation wavelength (in view of the spectral line width), often there is insufficient spectral spacing between the emission optima of different fluorescent molecules to permit individual and quantitative detection without substantial spectral overlap. Additionally, conventional fluorescent have limited fluorescence intensity. Further, currently available nonisotopic detection systems typically are limited in sensitivity due to the finite number of nonisotopic molecules which can be used to label a biomolecule to be detected.
Branched DNA or DNA dendrimers have been constructed as a signal amplification tool (see, e.g., U.S. Pat. Nos. 5,487,973, 5,484,904, and 5,175,270). These matrices are comprised of a DNA backbone having DNA arms. For example, matrices are constructed of successive subunits of a double-stranded DNA with single stranded arms on each end. Some of the arms are used to hybridize to a specific oligonucleotide probe, whereas other arms are used to bind to a nonisotopic or isotopic label. While providing for the addition of an relative increase in the number of label molecules as compared to other systems, one disadvantage is that a subunit needs to be custom-synthesized to contain at least one arm consisting of a complementary sequence which is capable of hybridizing to the specific DNA sequence which the user wishes to detect. Additionally, the label molecules are attached or ligated to the outwardly extending ends (tips) of the DNA matrices, rather than as an integral part of the matrix.
Thus, there remains a need for a nonisotopic detection system which (a) can result in generation of a signal comprising fluorescence emission of high quantum yield; (b) can result in signal amplification; (c) is not limited as to the chemical nature of the target molecule to be detected (e.g., versus detection of nucleic acid molecules only); (d) can be used to bind molecular probes of various types (versus binding to oligonucleotide probes only); (e) is preferably universal in terms of detecting target molecules of various sequences; and (f) can result in the simultaneous detection of more than one type of target molecule by utilizing a class of nonisotopic molecules that may be excited with a single excitation light source and with resultant fluorescence emissions with discrete fluorescence peaks.